Characterization of an ATP-dependent DNA ligase from the thermophilic archaeon Methanobacterium thermoautotrophicum

We report the production, purification and characterization of a DNA ligase encoded by the thermophilic archaeon Methanobacterium thermoautotrophicum. The 561 amino acid Mth ligase catalyzed strand-joining on a singly nicked DNA in the presence of a divalent cation (magnesium, manganese or cobalt) and ATP (K_m 1.1 µM). dATP can substitute for ATP, but CTP, GTP, UTP and NAD⁺ cannot. Mth ligase activity is thermophilic in vitro, with optimal nick-joining at 60°C. Mutational analysis of the conserved active site motif I (KxDG) illuminated essential roles for Lys251 and Asp253 at different steps of the ligation reaction. Mutant K251A is unable to form the covalent ligase–adenylate intermediate (step 1) and hence cannot seal a 3′-OH/5′-PO₄ nick. Yet, K251A catalyzes phosphodiester bond formation at a pre-adenylated nick (step 3). Mutant D253A is active in ligase–adenylate formation, but defective in activating the nick via formation of the DNA–adenylate intermediate (step 2). D253A is also impaired in phosphodiester bond formation at a pre-adenylated nick. A profound step 3 arrest, with accumulation of high levels of DNA–adenylate, could be elicited for the wild-type Mth ligase by inclusion of calcium as the divalent cation cofactor. Mth ligase sediments as a monomer in a glycerol gradient. Structure probing by limited proteolysis suggested that Mth ligase is a tightly folded protein punctuated by a surface-accessible loop between nucleotidyl transferase motifs III and IIIa.     

Kinetic mechanism and fidelity of nick sealing by Escherichia coli NAD+-dependent DNA ligase (LigA)

Escherichia coli DNA ligase (EcoLigA) repairs 3′-OH/5′-PO₄ nicks in duplex DNA via reaction of LigA with NAD⁺ to form a covalent LigA-(lysyl-Nζ)–AMP intermediate (step 1); transfer of AMP to the nick 5′-PO₄ to form an AppDNA intermediate (step 2); and attack of the nick 3′-OH on AppDNA to form a 3′-5′ phosphodiester (step 3). A distinctive feature of EcoLigA is its stimulation by ammonium ion. Here we used rapid mix-quench methods to analyze the kinetic mechanism of single-turnover nick sealing by EcoLigA–AMP. For substrates with correctly base-paired 3′-OH/5′-PO₄ nicks, k_step2 was fast (6.8–27 s⁻¹) and similar to k_step3 (8.3–42 s⁻¹). Absent ammonium, k_step2 and k_step3 were 48-fold and 16-fold slower, respectively. EcoLigA was exquisitely sensitive to 3′-OH base mispairs and 3′ N:abasic lesions, which elicited 1000- to >20000-fold decrements in k_step2. The exception was the non-canonical 3′ A:oxoG configuration, which EcoLigA accepted as correctly paired for rapid sealing. These results underscore: (i) how EcoLigA requires proper positioning of the nick 3′ nucleoside for catalysis of 5′ adenylylation; and (ii) EcoLigA''s potential to embed mutations during the repair of oxidative damage. EcoLigA was relatively tolerant of 5′-phosphate base mispairs and 5′ N:abasic lesions.     

Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi

The proteome of the amoebo-flagellate protozoan Naegleria gruberi is rich in candidate RNA repair enzymes, including 15 putative RNA ligases, one of which, NgrRnl, is a eukaryal homolog of Deinococcus radiodurans RNA ligase, DraRnl. Here we report that purified recombinant NgrRnl seals nicked 3′-OH/5′-PO₄ duplexes in which the 3′-OH strand is RNA. It does so via the “classic” ligase pathway, entailing reaction with ATP to form a covalent NgrRnl–AMP intermediate, transfer of AMP to the nick 5′-PO₄, and attack of the RNA 3′-OH on the adenylylated nick to form a 3′–5′ phosphodiester. Unlike members of the four known families of ATP-dependent RNA ligases, NgrRnl lacks a carboxy-terminal appendage to its nucleotidyltransferase domain. Instead, it contains a defining amino-terminal domain that we show is important for 3′-OH/5′-PO₄ nick-sealing and ligase adenylylation, but dispensable for phosphodiester synthesis at a preadenylylated nick. We propose that NgrRnl, DraRnl, and their homologs from diverse bacteria, viruses, and unicellular eukarya comprise a new “Rnl5 family” of nick-sealing ligases with a signature domain organization.     

Role of nucleotidyltransferase motifs I, III and IV in the catalysis of phosphodiester bond formation by Chlorella virus DNA ligase

ATP-dependent DNA ligases catalyze the sealing of 5′-phosphate and 3′-hydroxyl termini at DNA nicks by means of a series of three nucleotidyl transfer steps. Here we have analyzed by site-directed mutagenesis the roles of conserved amino acids of Chlorella virus DNA ligase during the third step of the ligation pathway, which entails reaction of the 3′-OH of the nick with the DNA–adenylate intermediate to form a phosphodiester and release AMP. We found that Asp65 and Glu67 in nucleotidyltransferase motif III and Glu161 in motif IV enhance the rate of step 3 phosphodiester formation by factors of 20, 1000 and 60, respectively. Asp29 and Arg32 in nucleotidyltransferase motif I enhance the rate of step 3 by 60-fold. Gel shift analysis showed that mutations of Arg32 and Asp65 suppressed ligase binding to a pre-adenylated nick, whereas Asp29, Glu67 and Glu161 mutants bound stably to DNA–adenylate. We infer that Asp29, Glu67 and Glu161 are involved directly in the step 3 reaction. In several cases, the effects of alanine or conservative mutations on step 3 were modest compared to their effects on the composite ligation reaction and individual upstream steps. These results, in concert with available crystallographic data, suggest that the active site of DNA ligase is remodeled during the three steps of the pathway and that some of the catalytic side chains play distinct roles at different stages.     

Kinetic mechanism of nick sealing by T4 RNA ligase 2 and effects of 3′-OH base mispairs and damaged base lesions

T4 RNA ligase 2 (Rnl2) repairs 3′-OH/5′-PO₄ nicks in duplex nucleic acids in which the broken 3′-OH strand is RNA. Ligation entails three chemical steps: reaction of Rnl2 with ATP to form a covalent Rnl2–(lysyl-Nζ)–AMP intermediate (step 1); transfer of AMP to the 5′-PO₄ of the nick to form an activated AppN– intermediate (step 2); and attack by the nick 3′-OH on the AppN– strand to form a 3′–5′ phosphodiester (step 3). Here we used rapid mix-quench methods to analyze the kinetic mechanism and fidelity of single-turnover nick sealing by Rnl2–AMP. For substrates with correctly base-paired 3′-OH nick termini, k_step2 was fast (9.5 to 17.9 sec⁻¹) and similar in magnitude to k_step3 (7.9 to 32 sec⁻¹). Rnl2 fidelity was enforced mainly at the level of step 2 catalysis, whereby 3′-OH base mispairs and oxoguanine, oxoadenine, or abasic lesions opposite the nick 3′-OH elicited severe decrements in the rate of 5′-adenylylation and relatively modest slowing of the rate of phosphodiester synthesis. The exception was the noncanonical A:oxoG base pair, which Rnl2 accepted as a correctly paired end for rapid sealing. These results underscore (1) how Rnl2 requires proper positioning of the 3′-terminal ribonucleoside at the nick for optimal 5′-adenylylation and (2) the potential for nick-sealing ligases to embed mutations during the repair of oxidative damage.     

Effects of 3′-OH and 5′-PO4 Base Mispairs and Damaged Base Lesions on the Fidelity of Nick Sealing by Deinococcus radiodurans RNA Ligase

Deinococcus radiodurans RNA ligase (DraRnl) is the founding member of a family of end-joining enzymes encoded by diverse microbes and viruses. DraRnl ligates 3′-OH, 5′-PO₄ nicks in double-stranded nucleic acids in which the nick 3′-OH end is RNA. Here we gauge the effects of 3′-OH and 5′-PO₄ base mispairs and damaged base lesions on the rate of nick sealing. DraRnl is indifferent to the identity of the 3′-OH nucleobase, provided that it is correctly paired. With 3′-OH mispairs, the DraRnl sealing rate varies widely, with G-T and A-C mispairs being the best substrates and G-G, G-A, and A-A mispairs being the worst. DraRnl accepts 3′ A–8-oxoguanine (oxoG) to be correctly paired, while it discriminates against U-oxoG and G-oxoG mispairs. DraRnl displays high activity and low fidelity in sealing 3′-OH ends opposite an 8-oxoadenine lesion. It prefers 3′-OH adenosine when sealing opposite an abasic template site. With 5′-PO₄ mispairs, DraRnl seals a 5′ T-G mispair as well as it does a 5′ C-G pair; in most other respects, the ligation fidelity at 5′ mispairs is similar to that at 3′ mispairs. DraRnl accepts a 5′ A-oxoG end to be correctly paired, yet it is more tolerant of 5′ T-oxoG and 5′ G-oxoG mispairs than the equivalent configurations on the 3′ side of the nick. At 5′ nucleobase-abasic site nicks, DraRnl prefers to ligate when the nucleobase is a purine. The biochemical properties of DraRnl are compatible with its participation in the templated repair of RNA damage or in the sealing of filled DNA gaps that have a 3′ ribopatch.     

Reprogramming the tRNA-splicing activity of a bacterial RNA repair enzyme

Programmed RNA breakage is an emerging theme underlying cellular responses to stress, virus infection and defense against foreign species. In many cases, site-specific cleavage of the target RNA generates 2′,3′ cyclic phosphate and 5′-OH ends. For the damage to be repaired, both broken ends must be healed before they can be sealed by a ligase. Healing entails hydrolysis of the 2′,3′ cyclic phosphate to form a 3′-OH and phosphorylation of the 5′-OH to form a 5′-PO₄. Here, we demonstrate that a polynucleotide kinase-phosphatase enzyme from Clostridium thermocellum (CthPnkp) can catalyze both of the end-healing steps of tRNA splicing in vitro. The route of tRNA repair by CthPnkp can be reprogrammed by a mutation in the 3′ end-healing domain (H189D) that yields a 2′-PO₄ product instead of a 2′-OH. Whereas tRNA ends healed by wild-type CthPnkp are readily sealed by T4 RNA ligase 1, the H189D enzyme generates ends that are spliced by yeast tRNA ligase. Our findings suggest that RNA repair enzymes can evolve their specificities to suit a particular pathway.     

Ligation reaction specificities of an NAD(+)-dependent DNA ligase from the hyperthermophile Aquifex aeolicus

An NAD⁺-dependent DNA ligase from the hyperthermophilic bacterium Aquifex aeolicus was cloned, expressed in Escherichia coli and purified to homogeneity. The enzyme is most active in slightly alkaline pH conditions with either Mg²⁺ or Mn²⁺ as the metal cofactor. Ca²⁺ and Ni²⁺ mainly support formation of DNA–adenylate intermediates. The catalytic cycle is characterized by a low k_cat value of 2 min^–1 with concomitant accumulation of the DNA–adenylate intermediate when Mg²⁺ is used as the metal cofactor. The ligation rates of matched substrates vary by up to 4-fold, but exhibit a general trend of T/A ≤ G/C < C/G < A/T on both the 3′- and 5′-side of the nick. Consistent with previous studies on Thermus ligases, this Aquifex ligase exhibits greater discrimination against a mismatched base pair on the 3′-side of the nick junction. The requirement of 3′ complementarity for a ligation reaction is reaffirmed by results from 1 nt insertions on either the 3′- or 5′-side of the nick. Furthermore, most of the unligatable 3′ mismatched base pairs prohibit formation of the DNA–adenylate intermediate, indicating that the substrate adenylation step is also a control point for ligation fidelity. Unlike previously studied ATP ligases, gapped substrates cannot be ligated and intermediate accumulation is minimal, suggesting that complete elimination of base pair complementarity on one side of the nick affects substrate adenylation on the 5′-side of the nick junction. Relationships among metal cofactors, ligation products and intermediate, and ligation fidelity are discussed.     

DNA3′pp5′G de-capping activity of aprataxin: effect of cap nucleoside analogs and structural basis for guanosine recognition

DNA_3′pp_5′G caps synthesized by the 3′-PO₄/5′-OH ligase RtcB have a strong impact on enzymatic reactions at DNA 3′-OH ends. Aprataxin, an enzyme that repairs A_5′pp_5′DNA ends formed during abortive ligation by classic 3′-OH/5′-PO₄ ligases, is also a DNA 3′ de-capping enzyme, converting DNAppG to DNA_3′p and GMP. By taking advantage of RtcB''s ability to utilize certain GTP analogs to synthesize DNAppN caps, we show that aprataxin hydrolyzes inosine and 6-O-methylguanosine caps, but is not adept at removing a deoxyguanosine cap. We report a 1.5 Å crystal structure of aprataxin in a complex with GMP, which reveals that: (i) GMP binds at the same position and in the same anti nucleoside conformation as AMP; and (ii) aprataxin makes more extensive nucleobase contacts with guanine than with adenine, via a hydrogen bonding network to the guanine O6, N1, N2 base edge. Alanine mutations of catalytic residues His147 and His149 abolish DNAppG de-capping activity, suggesting that the 3′ de-guanylylation and 5′ de-adenylylation reactions follow the same pathway of nucleotidyl transfer through a covalent aprataxin-(His147)–NMP intermediate. Alanine mutation of Asp63, which coordinates the guanosine ribose hydroxyls, impairs DNAppG de-capping.     

A kinetic framework for tRNA ligase and enforcement of a 2′-phosphate requirement for ligation highlights the design logic of an RNA repair machine

tRNA ligases are essential components of informational and stress-response pathways entailing repair of RNA breaks with 2′,3′-cyclic phosphate and 5′-OH ends. Plant and fungal tRNA ligases comprise three catalytic domains. Phosphodiesterase and kinase modules heal the broken ends to generate the 3′-OH, 2′-PO₄, and 5′-PO₄ required for sealing by the ligase. We exploit RNA substrates with different termini to define rates of individual steps or subsets of steps along the repair pathway of plant ligase AtRNL. The results highlight rate-limiting transactions, how repair is affected by active-site mutations, and how mutations are bypassed by RNA alterations. We gain insights to 2′-PO₄ specificity by showing that AtRNL is deficient in transferring AMP to pRNA_OH to form AppRNA_OH but proficient at sealing pre-adenylylated AppRNA_OH. This strategy for discriminating 2′-PO₄ versus 2′-OH ends provides a quality-control checkpoint to ensure that only purposeful RNA breaks are sealed and to avoid nonspecific “capping” of 5′-PO₄ ends.     

Deinococcus radiodurans RNA ligase exemplifies a novel ligase clade with a distinctive N-terminal module that is important for 5'-PO4 nick sealing and ligase adenylylation but dispensable for phosphodiester formation at an adenylylated nick

Deinococcus radiodurans RNA ligase (DraRnl) is a template-directed ligase that seals nicked duplexes in which the 3′-OH strand is RNA. DraRnl is a 342 amino acid polypeptide composed of a C-terminal adenylyltransferase domain fused to a distinctive 126 amino acid N-terminal module (a putative OB-fold). An alanine scan of the C domain identified 9 amino acids essential for nick ligation, which are located within nucleotidyltransferase motifs I, Ia, III, IIIa, IV and V. Seven mutants were dysfunctional by virtue of defects in ligase adenylylation: T163A, H167A, G168A, K186A, E230A, F281A and E305A. Four of these were also defective in phosphodiester formation at a preadenylylated nick: G168A, E230A, F281A and E305A. Two nick sealing-defective mutants were active in ligase adenylylation and sealing a preadenylylated nick, thereby implicating Ser185 and Lys326 in transfer of AMP from the enzyme to the nick 5′-PO₄. Whereas deletion of the N-terminal domain suppressed overall nick ligation and ligase adenylylation, it did not compromise sealing at a preadenylylated nick. Mutational analysis of 15 residues of the N domain identified Lys26, Gln31 and Arg79 as key constituents. Structure–activity relationships at the essential residues were determined via conservative substitutions. We propose that DraRnl typifies a new clade of polynucleotide ligases. DraRnl homologs are detected in several eukaryal proteomes.     

Distinctive kinetics and substrate specificities of plant and fungal tRNA ligases

Plant and fungal tRNA ligases are trifunctional enzymes that repair RNA breaks with 2′,3′-cyclic-PO₄ and 5′-OH ends. They are composed of cyclic phosphodiesterase (CPDase) and polynucleotide kinase domains that heal the broken ends to generate the 3′-OH, 2′-PO₄, and 5′-PO₄ required for sealing by a ligase domain. Here, we use short _HORNA>p substrates to determine, in a one-pot assay format under single-turnover conditions, the order and rates of the CPDase, kinase and ligase steps. The observed reaction sequence for the plant tRNA ligase AtRNL, independent of RNA length, is that the CPDase engages first, converting _HORNA>p to _HORNA_2′p, which is then phosphorylated to pRNA_2′p by the kinase. Whereas the rates of the AtRNL CPDase and kinase reactions are insensitive to RNA length, the rate of the ligase reaction is slowed by a factor of 16 in the transition from 10-mer RNA to 8-mer and further by eightfold in the transition from 8-mer RNA to 6-mer. We report that a single ribonucleoside-2′,3′-cyclic-PO₄ moiety enables AtRNL to efficiently splice an otherwise all-DNA strand. Our characterization of a fungal tRNA ligase (KlaTrl1) highlights important functional distinctions vis à vis the plant homolog. We find that (1) the KlaTrl1 kinase is 300-fold faster than the AtRNL kinase; and (2) the KlaTrl1 kinase is highly specific for GTP or dGTP as the phosphate donor. Our findings recommend tRNA ligase as a tool to map ribonucleotides embedded in DNA and as a target for antifungal drug discovery.     

Effects of DNA3′pp5′G capping on 3′ end repair reactions and of an embedded pyrophosphate-linked guanylate on ribonucleotide surveillance

When DNA breakage results in a 3′-PO₄ terminus, the end is considered ‘dirty’ because it cannot prime repair synthesis by DNA polymerases or sealing by classic DNA ligases. The noncanonical ligase RtcB can guanylylate the DNA 3′-PO₄ to form a DNA_3′pp_5′G_OH cap. Here we show that DNA capping precludes end joining by classic ATP-dependent and NAD⁺-dependent DNA ligases, prevents template-independent nucleotide addition by mammalian terminal transferase, blocks exonucleolytic proofreading by Escherichia coli DNA polymerase II and inhibits proofreading by E. coli DNA polymerase III, while permitting templated DNA synthesis from the cap guanosine 3′-OH primer by E. coli DNA polymerase II (B family) and E. coli DNA polymerase III (C family). Human DNA polymerase β (X family) extends the cap primer predominantly by a single templated addition step. Cap-primed synthesis by templated polymerases embeds a pyrophosphate-linked ribonucleotide in DNA. We find that the embedded ppG is refractory to surveillance and incision by RNase H2.     

Structure-function analysis of the kinase-CPD domain of yeast tRNA ligase (Trl1) and requirements for complementation of tRNA splicing by a plant Trl1 homolog

Trl1 is an essential 827 amino acid enzyme that executes the end-healing and end-sealing steps of tRNA splicing in Saccharomyces cerevisiae. Trl1 consists of two domains—an N-terminal ligase component and a C-terminal 5′-kinase/2′,3′-cyclic phosphodiesterase (CPD) component—that can function in tRNA splicing in vivo when expressed as separate polypeptides. To understand the structural requirements for the kinase-CPD domain, we performed an alanine scan of 30 amino acids that are conserved in Trl1 homologs from other fungi. We thereby identified four residues (Arg463, His515, Thr675 and Glu741) as essential for activity in vivo. Structure–function relationships at these positions, and at four essential or conditionally essential residues defined previously (Asp425, Arg511, His673 and His777), were clarified by introducing conservative substitutions. Biochemical analysis showed that lethal mutations of Asp425, Arg463, Arg511 and His515 in the kinase module abolished polynucleotide kinase activity in vitro. We report that a recently cloned 1104 amino acid Arabidopsis RNA ligase functions in lieu of yeast Trl1 in vivo and identify essential side chains in the ligase, kinase and CPD modules of the plant enzyme. The plant ligase, like yeast Trl1 but unlike T4 RNA ligase 1, requires a 2′-PO₄ end for tRNA splicing in vivo.     

Pseudomonas putida MPE,a manganese-dependent endonuclease of the binuclear metallophosphoesterase superfamily,incises single-strand DNA in two orientations to yield a mixture of 3′-PO4 and 3′-OH termini

Pseudomonas putida MPE exemplifies a novel clade of manganese-dependent single-strand DNA endonuclease within the binuclear metallophosphoesterase superfamily. MPE is encoded within a widely conserved DNA repair operon. Via structure-guided mutagenesis, we identify His113 and His81 as essential for DNA nuclease activity, albeit inessential for hydrolysis of bis-p-nitrophenylphosphate. We propose that His113 contacts the scissile phosphodiester and serves as a general acid catalyst to expel the OH leaving group of the product strand. We find that MPE cleaves the 3′ and 5′ single-strands of tailed duplex DNAs and that MPE can sense and incise duplexes at sites of short mismatch bulges and opposite a nick. We show that MPE is an ambidextrous phosphodiesterase capable of hydrolyzing the ssDNA backbone in either orientation to generate a mixture of 3′-OH and 3′-PO₄ cleavage products. The directionality of phosphodiester hydrolysis is dictated by the orientation of the water nucleophile vis-à-vis the OH leaving group, which must be near apical for the reaction to proceed. We propose that the MPE active site and metal-bound water nucleophile are invariant and the enzyme can bind the ssDNA productively in opposite orientations.     

Characterization of a Baculovirus-Encoded ATP-Dependent DNA Ligase

Sequence analysis of the Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV) genome identified an open reading frame (ORF) encoding a 548-amino-acid (62-kDa) protein that showed 35% amino acid sequence identity with vaccinia virus ATP-dependent DNA ligase. Ligase homologs have not been reported from other baculoviruses. The ligase ORF was cloned and expressed as an N-terminal histidine-tagged fusion protein. Incubation of the purified protein with [α-³²P]ATP resulted in formation of a covalent enzyme-adenylate intermediate which ran as a 62-kDa labeled band on a sodium dodecyl sulfate-polyacrylamide gel. Loss of the radiolabeled band occurred upon incubation of the intermediate with pyrophosphate, poly(dA) · poly(dT)_12–18, or poly(rA) · poly(dT)_12–18, characteristics of a DNA ligase II or III. The protein was able to ligate a double-stranded synthetic DNA substrate containing a single nick and inefficiently ligated a 1-nucleotide (nt) gap but did not ligate a 2-nt gap. It was able to ligate short, complementary overhangs but not blunt-ended double-stranded DNA. In a transient DNA replication assay employing six plasmids containing the LdMNPV homologs of the essential baculovirus replication genes, a plasmid containing the DNA ligase gene was neither essential nor stimulatory. All of these results are consistent with the activity of type III DNA ligases, which have been implicated in DNA repair and recombination.     

Characterization of Agrobacterium tumefaciens DNA ligases C and D

Agrobacterium tumefaciens encodes a single NAD⁺-dependent DNA ligase and six putative ATP-dependent ligases. Two of the ligases are homologs of LigD, a bacterial enzyme that catalyzes end-healing and end-sealing steps during nonhomologous end joining (NHEJ). Agrobacterium LigD1 and AtuLigD2 are composed of a central ligase domain fused to a C-terminal polymerase-like (POL) domain and an N-terminal 3′-phosphoesterase (PE) module. Both LigD proteins seal DNA nicks, albeit inefficiently. The LigD2 POL domain adds ribonucleotides or deoxyribonucleotides to a DNA primer-template, with rNTPs being the preferred substrates. The LigD1 POL domain has no detectable polymerase activity. The PE domains catalyze metal-dependent phosphodiesterase and phosphomonoesterase reactions at a primer-template with a 3′-terminal diribonucleotide to yield a primer-template with a monoribonucleotide 3′-OH end. The PE domains also have a 3′-phosphatase activity on an all-DNA primer-template that yields a 3′-OH DNA end. Agrobacterium ligases C2 and C3 are composed of a minimal ligase core domain, analogous to Mycobacterium LigC (another NHEJ ligase), and they display feeble nick-sealing activity. Ligation at DNA double-strand breaks in vitro by LigD2, LigC2 and LigC3 is stimulated by bacterial Ku, consistent with their proposed function in NHEJ.     

Structures of Bacterial Polynucleotide Kinase in a Michaelis Complex with Nucleoside Triphosphate (NTP)-Mg2+ and 5′-OH RNA and a Mixed Substrate-Product Complex with NTP-Mg2+ and a 5′-Phosphorylated Oligonucleotide

Clostridium thermocellum polynucleotide kinase (CthPnk), the 5′-end-healing module of a bacterial RNA repair system, catalyzes reversible phosphoryl transfer from a nucleoside triphosphate (NTP) donor to a 5′-OH polynucleotide acceptor, either DNA or RNA. Here we report the 1.5-Å crystal structure of CthPnk-D38N in a Michaelis complex with GTP-Mg²⁺ and a 5′-OH RNA oligonucleotide. The RNA-binding mode of CthPnk is different from that of the metazoan RNA kinase Clp1. CthPnk makes hydrogen bonds to the ribose 2′-hydroxyls of the 5′ terminal nucleoside, via Gln51, and the penultimate nucleoside, via Gln83. The 5′-terminal nucleobase is sandwiched by Gln51 and Val129. Mutating Gln51 or Val129 to alanine reduced kinase specific activity 3-fold. Ser37 and Thr80 donate functionally redundant hydrogen bonds to the terminal phosphodiester; a S37A-T80A double mutation reduced kinase activity 50-fold. Crystallization of catalytically active CthPnk with GTP-Mg²⁺ and a 5′-OH DNA yielded a mixed substrate-product complex with GTP-Mg²⁺ and 5′-PO₄ DNA, wherein the product 5′ phosphate group is displaced by the NTP γ phosphate and the local architecture of the acceptor site is perturbed.     

Solution NMR Studies of Chlorella Virus DNA Ligase-adenylate

DNA ligases are essential guardians of genome integrity by virtue of their ability to recognize and seal 3′-OH/5′-phosphate nicks in duplex DNA. The substrate binding and three chemical steps of the ligation pathway are coupled to global and local changes in ligase structure, involving both massive protein domain movements and subtle remodeling of atomic contacts in the active site. Here we applied solution NMR spectroscopy to study the conformational dynamics of the Chlorella virus DNA ligase (ChVLig), a minimized eukaryal ATP-dependent ligase consisting of nucleotidyltransferase, OB, and latch domains. Our analysis of backbone ¹⁵N spin relaxation and ¹⁵N,¹H residual dipolar couplings of the covalent ChVLig-AMP intermediate revealed conformational sampling on fast (picosecond to nanosecond) and slow timescales (microsecond to millisecond), indicative of interdomain and intradomain flexibility. We identified local and global changes in ChVLig-AMP structure and dynamics induced by phosphate. In particular, the chemical shift perturbations elicited by phosphate were clustered in the peptide motifs that comprise the active site. We hypothesize that phosphate anion mimics some of the conformational transitions that occur when ligase-adenylate interacts with the nick 5′-phosphate.     

Mutational analysis defines the 5'-kinase and 3'-phosphatase active sites of T4 polynucleotide kinase

T4 polynucleotide kinase (Pnk) is a bifunctional 5′-kinase/3′-phosphatase that aids in the repair of broken termini in RNA by converting 3′-PO₄/5′-OH ends into 3′-OH/5′-PO₄ ends, which are then sealed by RNA ligase. Here we have employed site-directed mutagenesis (introducing 31 mutations at 16 positions) to locate candidate catalytic residues within the 301 amino acid Pnk polypeptide. We found that alanine substitutions for Arg38 and Arg126 inactivated the 5′-kinase, but spared the 3′-phosphatase activity. Conservative substitutions of lysine or glutamine for Arg38 and Arg126 did not restore 5′-kinase activity. These results, together with previous mutational studies, highlight a constellation of five amino acids (Lys15, Ser16, Asp35, Arg38 and Arg126) that likely comprise the 5′-kinase active site. Four of these residues are conserved at the active sites of adenylate kinases (Adk), suggesting that Pnk and Adk are structurally and mechanistically related. We found that alanine substitutions for Asp165, Asp167, Arg176, Arg213, Asp254 and Asp278 inactivated the 3′-phosphatase, but spared the 5′-kinase. Conservative substitutions of asparagine or glutamate for Asp165, Asp167 and Asp254 did not revive the 3′-phosphatase activity, nor did lysine substitutions for Arg176 and Arg213. Glutamate in lieu of Asp278 partially restored activity, whereas asparagine had no salutary effect. Alanine substitutions for Arg246 and Arg279 partially inactivated the 3′-phosphatase; the conservative R246K change restored activity, whereas R279K had no benefit. The essential phosphatase residues Asp165 and Asp167 are located within a ¹⁶⁵DxDxT¹⁶⁹ motif that defines a superfamily of phosphotransferases. Our data suggest that the 3′-phosphatase active site incorporates multiple additional functional groups.